Sex determination by simultaneous application of polymerase chain reaction and fluorescent in situ hybridization on the same blastomere of a pre-embryo.

OBJECTIVE: To describe an investigation of the feasibility of carrying out polymerase chain reaction (PCR) and fluorescent in situ hybridization on the same single human cell for sex determination. DESIGN: To develop protocol for a clinical diagnostic test in preclinical trials. SETTING: Infertile human volunteers in a clinical environment. PATIENTS: Polyploid embryos were obtained from patients treated by IVF at the Advanced Institute of Fertility. INTERVENTIONS: Seventeen blastomeres biopsied from human polyploid embryos were analyzed for sex determination by simultaneous application of PCR and fluorescent in situ hybridization. MAIN OUTCOME MEASURE: Presence of an X- or X and Y-chromosome band after agarose gel electrophoresis of PCR products and presence of an X- or X and Y-chromosome fluorescent probe signal after fluorescent in situ hybridization following PCR analysis. RESULTS: By PCR, all 17 blastomeres were amplified and, by fluorescent in situ hybridization, 12 (70.6%) blastomeres produced signals that were consistent with PCR results. Two blastomeres showed only X signals, although by PCR they had both X and Y-chromosome bands. CONCLUSIONS: The sequential use of PCR and fluorescent in situ hybridization on the same blastomere can be applied to improve the accuracy of sex determination before fresh ET.

[Quality control and cytogenetic analysis of human pre-embryos fertilized and cultured in vitro].

For the purpose to evaluate in vitro culture conditions of human preembryos, the efficacy of conventional culture and co-culture systems on embryonic development and genetic disorders was studied. Firstly, the development of cultured mouse embryos grown in standard media (Whitten's, GPM, HFT and Ham F10) or in HFT medium with different helper cell layers was compared. Embryonic growth was substantially reduced during in vitro culture, demonstrably by impaired cell proliferation, compared with in vivo controls. In in vitro fertilization and culture condition, SCEs of blastocysts were significantly increased. Development in co-culture with the feeder layers was notably better than in standard media. These results suggest that human preembryos could be rescued by the use of helper cells. Increased developmental rates and the cell numbers of blastocysts were the most evident morphological features of human preembryos that developed in co-culture with uterine luminal epithelial cells. However mosaicism may be caused by in vitro culture conditions and its onset may indicate when a disturbance in the embryonic development has occurred. It is advisable to perform further research into the mechanism of feeder cell-embryo interaction for understanding the optimal conditions of embryonic development in vitro.

[Biopsy of mouse embryo fertilized in vitro as a preclinical model for preimplantation genetic diagnosis].

In preimplantation genetic diagnosis, it is vital that the biopsy method does not affect embryonic development and yet provides a suitable specimen for genetic diagnosis. This study investigated whether the expulsion method, a modification of the displacement and extrusion methods, could satisfy the above mentioned conditions. Mouse embryos fertilized in vitro were biopsied at the 4, 8 and 16-cell stages, and were subsequently observed for in vitro and in vivo development. The rates of blastocyst formation, implantation, and fetal growth in the 8- and 16-cell biopsy groups were not significantly different from those in the control groups, but all 3 parameters were significantly reduced in the 4-cell group. Fetal and placental development was similar to the control group when embryos were biopsied at all three stages. Single blastomeres obtained by expulsion biopsy were subjected to DNA amplification by dual PCR, and Sry and myogenin sequences were amplified. In conclusion, expulsion biopsy did not affect the development of 8- and 16-cell embryos, and the specimens obtained were adequate for DNA amplification.

Preimplantation diagnosis by fluorescence in situ hybridization using 13-, 16-, 18-, 21-, 22-, X-, and Y-chromosome probes.

PURPOSE: Our purpose was to select the proper chromosomes for preimplantation diagnosis based on aneuploidy distribution in abortuses and to carry out a feasibility study of preimplantation diagnosis for embryos using multiple-probe fluorescence in situ hybridization (FISH) on the selected chromosomes of biopsied blastomeres. METHODS: After determining the frequency distribution of aneuploidy found in abortuses, seven chromosomes were selected for FISH probes. Blastomeres were obtained from 33 abnormal or excess embryos. The chromosome complements of both the biopsied blastomeres and the remaining sibling blastomeres in each embryo were determined by FISH and compared to evaluate their preimplantation diagnostic potential. RESULTS: Chromosomes (16, 22, X, Y) and (13, 18, 21) were selected on the basis of the high aneuploid prevalence in abortuses for the former group and the presence of trisomy in the newborn for the latter. Thirty-six (72%) of 50 blastomeres gave signals to permit a diagnosis. Diagnoses made from biopsied blastomeres were consistent with the diagnoses made from the remaining sibling blastomeres in 18 embryos. In only 2 of 20 cases did the biopsied blastomere diagnosis and the embryo diagnosis not match. CONCLUSIONS: If FISH of biopsied blastomere was successful, a preimplantation diagnosis could be made with 10% error. When a combination of chromosome-13, -16, -18, -21, -22, -X, and -Y probes was used, up to 65% of the embryos destined to be aborted could be detected.